Microscope

ABSTRACT

A confocal microscope has a specimen holding device for holding a specimen ( 18 ). The specimen ( 18 ) is illuminated by an illuminating unit ( 10 ). An optics unit ( 12 ) serves to direct radiation produced by the illuminating unit ( 10 ) toward the specimen ( 18 ) and to direct the radiation emitted by the specimen toward a detector unit ( 20 ). The confocal microscope also comprises an aperture diaphragm ( 34 ) that is placed in the beam path in front of the detector unit ( 20 ). In addition, a focusing lens ( 30 ) is provided in the beam path in front of the aperture diaphragm ( 34 ). The focusing lens ( 30 ) can be moved in order to adjust the confocal microscope, for example, in order to compensate for thermal stresses.

This application is a national stage application in the U.S. ofInternational Patent Application No. PCT/EP02/01610, filed Feb. 15,2002, and which claims priority from German Patent Application No. 10107 210.4, filed Feb. 16, 2001.

FIELD OF THE INVENTION

The invention relates to a microscope, particularly a confocalmicroscope for the single-molecule spectroscopy. In the following, anykind of magnifying means composed of two lens arrangements will beunderstood by a microscope.

BACKGROUND OF THE INVENTION

Confocal microscopes are particularly suitable for the high-resolutionmeasurement in the direction of the Z-axis, i.e., in longitudinaldirections of the beam path of the microscope. To this end, confocalmicroscopes comprise an illuminating unit that typically is a laser. Aspecimen held by the specimen holding device is illuminated by theilluminating unit. To this end, an optics unit is provided which directsradiation produced by the illuminating unit toward the specimen anddirects radiation given off by the specimen toward a detector unit. As apart of the optics, conventional confocal microscopes comprise a beamsplitter. On the one hand, the beam splitter directs the light emittedfrom the illuminating unit toward the specimen and on the other hand, itleaves through the light reflected by the specimen so that it can reacha detector unit arranged behind the beam splitter in the beam paththrough an aperture diaphragm. As an illuminating unit, differentilluminating units may be provided which, for example, produce visiblelight or also wavelengths in the non-visible range. In front of thedetector unit, an aperture diaphragm is arranged and a focusing lens isarranged in front of the latter.

By means of the focusing lens, the beam path is focused in the openingof the aperture diaphragm.

By focusing the beam path in the opening of the aperture diaphragm, arelatively high light flux reaches the detector. Even a slightdefocusing by displacing the object plane in Z-direction leads to ablurred image on the aperture diaphragm. This results in a smaller lightflux since the focus of the focusing lens is no longer focused in theopening of the aperture diaphragm and thus, a smaller light flux comesthrough the diaphragm opening. Displacing the object plane in theX-Y-plane also results in a displacement of the focus in the plane ofthe aperture diaphragm. Thereby, the light quantity passing the aperturediaphragm is reduced since the focus is no longer focused in the openingof the aperture diaphragm.

When confocal microscopes are used in high-throughput screening, thefocus of the microscope objective is arranged in a biological orchemical specimen. Since the specimens are minimum quantities ofspecimen liquid having a volume in the microliter or nanoliter range,the confocal microscope used in high-throughput screening must be ahighly precise device. This requirement exists all the more as specimensin the submicroliter range are examined in modern high-throughputscreening installations.

Because of the required very high accuracy of the focusing in thespecimen, even very small temperature changes lead to the maladjustmentof the confocal microscope. Particularly, a temperature-dependentmaladjustment of the aperture diaphragm itself leads to the impairmentof the accuracy of the microscope. Even slight maladjustments lead tothat the illumination-side and the detection-side focus are no longercongruent. This results in a signal displacement and a considerablefalsification of the measuring results. In addition, in thesingle-molecule detection, the assumption of the focus geometry is nolonger valid in case of even a slight maladjustment. Further, theaccuracy of the measuring results is influenced by inaccuracies of thelaser by which the focus is displaced as well. Further, the measuringaccuracy of confocal microscopes is influenced by the fact that the beamsplitter provided in the optics unit has to be exchanged in dependenceon the wavelength produced by the laser and given off by the specimen.Upon exchanging the beam splitter, slight position changes thereofoccur. This also leads to a focus displacement and thus to afalsification of the measuring results. The beam splitter can displacerelative to the excitation optics and the objective by temperatureinfluences.

For adjusting, it is known from U.S. Pat. No. 4,863,226 to provide anadjusting mechanism for the aperture diaphragm. By the adjustingmechanism, the aperture diaphragm can be displaced in the direction ofthe X-Y- and Z-axis. Since the distance and the leading position betweenthe aperture diaphragm and the detector arranged behind the aperturediaphragm have to be observed very closely, it is required to displacethe entire detector unit together with the aperture diaphragm.Particularly with modern confocal microscopes, very complicated andsensitive detectors such as photo multiplier or spectographicmultidetector arrangements are used. This results in that the detectorunit occupies a large building space and is heavy. Therefore, theaccurate positioning of the aperture diaphragm in the direction of thethree axes is accompanied with considerable mechanical efforts. In thisconnection, it has to be considered that the adjustment of the aperturediaphragm has to be effected in the micrometer range.

From U.S. Pat. No. 5,334,830, it is further known to arrange additionaladjustable tilted mirrors for adjusting the aperture diaphragm in thebeam path. Aligning the focus with the opening of the aperture diaphragmis thus effected by adjusting the tilted mirrors arranged in the beampath. By arranging additional components such as transparent camerawedges in the beam path, color errors as well as reflection lossesoccur. Further, the structural length of the microscope increases.

It is the object of the invention to provide a microscope which is welladjustable with as small mechanical efforts as possible, particularly incase of highly precise requirements.

SUMMARY OF THE INVENTION

This object is solved, according to the invention, by the features ofthe following embodiments respectively. In one embodiment of the presentinvention, a microscope, particularly a confocal microscope, for thesingle-molecule spectroscopy is provided that includes: a specimenholding device for holding a specimen, an illuminating unit, an opticsunit directing radiation produced by the illuminating unit towards thespecimen and directing radiation given off by the specimen toward adetector unit, an aperture diaphragm arranged in the beam path in frontof the detector unit, and a focusing lens arranged in the beam path infront of the aperture diaphragm, characterized in that the focusing lensis vertically adjustable with respect to its optical axis. In anotherembodiment of the present invention, a microscope, particularly confocalmicroscope, for the single-molecule spectroscopy, is provided thatincludes: a specimen holding device for holding a specimen, anilluminating unit, an optics unit comprising a beam splitter, whichdirects radiation produced by the illuminating unit towards the specimenand radiation given off by the specimen toward a detector unit, anaperture diaphragm arranged in the beam path in front of the detectorunit, and a stationary focusing lens arranged in the beam path in frontof the aperture diaphragm, characterized in that the beam splitter isadapted to be tilted to align the radiation given off by the specimenwith the central axis of the opening of the aperture diaphragm.

BRIEF DESCRIPTION OF THE DRAWING

Hereinafter, the invention is explained in detail with respect to apreferred embodiment with reference to the appended drawing. The drawingshows a schematic structure of a confocal microscope according to theinvention.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE INVENTION

According to the invention, adjusting is effected either by adjustingthe focusing lens through which the radiation given off by the specimenis fixed onto the aperture diaphragm or by adjusting a beam splitterthat may be provided in the optics unit. According to the invention, theaperture diaphragm is thus at most displaced in Z-direction, i.e., inthe direction of its optical axis. Preferably, the aperture diaphragm isstationary.

The structure of the microscope according to the invention has theadvantage that the adjustment of the aperture diaphragm together withthe detector unit, which is possible with considerable mechanicalefforts, is avoided in X-, Y- and Z-direction. The efforts areconsiderably reduced already when the aperture diaphragm, together withthe detector unit, is exclusively adjustable in the direction of theZ-axis. Mechanically, the adjustment of the confocal microscope byadjusting the focusing lens and/or the beam splitter can be realizedmuch more easily.

Experiments have shown that in contrast to the prevailing opinion, theoptical errors occurring by adjusting the focusing lens and/or the beamsplitter are negligibly small.

This also applies to confocal microscopes in particular. In thisconnection, it has to be guaranteed by the confocal microscope that adiffraction-limited measuring volume as small as possible is producedand observed. To this end, an objective with a numerical aperture ashigh as possible is used which is completely filled up by theillumination light. Thereby, the radius of the illuminated focus isdefined. The size of the observed focus is adapted to this radius. Witha given focus of the focus lens which amounts to between 160 and 200 mmin typical microscope arrangements, this adaptation can be achieved byselecting the diameter of the aperture diaphragm. To avoid significantlosses of detected light as well as asymmetries of the measuring volume,the focus on the aperture diaphragm is only allowed to laterally shiftby fractions of the diaphragm diameter. In case of confocal microscopeswith high resolution, for example, this results in an objective focus off=4 mm and a numerical aperture of 1.2. In case of a focus of the tubelens of 180 mm, the diameter of the aperture diaphragm amounts to about30 μm. This results in an admissible shift of the focus in the X-Y-planeof 5 μm at most. An admissible angle error of 6″ corresponds thereto.

In a first embodiment of the invention, the focusing lens is thusvertically adjustable to its optical axis, i.e., in the direction of theX- and/or Y-axis. Adjusting in the direction of the respectively otheraxis in the X-Y-plane can be effected by tilting the beam splitter.Particularly in case of confocal microscopes without a beam splitter orwhere the beam splitter cannot be exchanged, for example, and is thuspreferably fixed in an non-adjustable position, the focusing withrespect to the X-Y-plane is exclusively effected by displacing thefocusing lens within this plane.

In this embodiment, the adjustment in the direction of the Z-axis can beeffected by displacing the aperture diaphragm together with the detectorunit. But it is also particularly preferred to realize the adjustment inthe direction of the Z-axis by displacing the focusing lens in thedirection of its optical axis, i.e., in the direction of the Z-axis.

The adjustment in the X-Y-plane is further possible by adjusting thebeam splitter, preferably by tilting the beam splitter. Thereby, thebeam path can be aligned with the central axis of the opening of theaperture diaphragm. The beam splitter can be adjusted in addition to theadjustment possibilities of the focusing lens.

In another preferred embodiment of the invention, only the beam splitteris adapted to be tilted. By tilting the beam splitter, an adjustment inthe X-Y-plane can be effected. In this embodiment, the focusing lens isstationary.

For the adjustment in the Z-direction, the aperture diaphragm isdisplaceable together with the detector unit. The use of confocalmicroscopes in high-throughput screening requires the satisfaction ofspecial requirements in order to increase the throughput, on the onehand, i.e. to reduce the measuring times of any single specimen, ifpossible. Therefore, the efficiency of the fluorescent light through thedetector has to be optimized. This means that the numerical aperture ofthe detection optics the transmission of all the optical components inthe detection path, at least in the relevant spectral region, as well asthe quantum yield of the detector have to be optimized. In this respect,the microscope according to the invention has the advantage that theadjustment of the detection optics is effected without any additionalcomponents such as a tilted mirror or a camera wedge. This has theadvantage that optical losses occurring because of these additionalcomponents do not occur.

Further, such microscopes are often used in high-throughput screening tobe able to analyze several parameters, the intensity at differentwavelengths or the polarization directions, for example. Since themeasuring results are impaired when the fluorescence decays, theseparameters should be measured simultaneously, if possible. This resultsin an extremely complicated detector structure. Since, according to theinvention, the detector unit need not be moved or only in a particulardirection together with the aperture diaphragm, the microscope accordingto the invention has a considerable advantage over existing microscopessince particularly the movement of large masses in narrow ranges oftolerance is not required.

Because of the afore-described advantages, the microscope according tothe invention is particularly suitable for the single-moleculedetection. The single-molecule detection is particularly suitable forthe high-throughput screening since the measurement can be carried outwith small substance amounts. Particularly, the afore-describedadvantages have a particularly advantageous effect since the individualmolecules only emit weak signals. The impairment of weak signals byadditional optical elements leads to a considerable falsification of themeasuring results.

Further, there is the requirement, particularly for confocalmicroscopes, that the overlapping of the illumination and detection focicorresponds. In order to realize this, an as symmetrical and constantgeometry as possible is required over the entire screening duration.This has its roots in that model assumptions concerning the measuringvolume geometry enter such into the evaluation of the measurement andthus into the determination of the required single molecule properties.This particularly results in the requirement of stability of themeasuring arrangement over the time and the possibility of a regular,possibly automatic, adjustment of the detection optics. This is possiblewith the microscope according to the invention.

The opening of the aperture diaphragm, for example, may be circular orsplit-shaped. Particularly in case of a split-shaped opening of theaperture diaphragm, it is advantageous to permit a rotation of theaperture diaphragm about its central axis. Thereby, the split-shapedopening of the aperture diaphragm can be aligned by simply rotating theaperture diaphragm.

An automatic adjustment of the microscope is particularly preferred. Tothis end, the focusing lens and/or the beam splitter comprises anadjusting mechanism permitting an adjustment of the focusing lens and/orthe beam splitter via suitable drive elements, e.g., step motors or D.C.motor with position encoder. Additionally, an adjusting mechanism fordisplacing the aperture diaphragm together with the detector unit in thedirection of the Z-axis may be provided. Preferably, the adjustingtolerance with respect to the opening of the aperture diaphragm issmaller than 3 μm, particularly preferably smaller than 2 μm.

As an illuminating unit, the confocal microscope comprises a laser 10.The light emitted by the laser 10 is directed onto a specimen 18arranged in a recess 14 of a specimen support 16, such as a titer plate,by means of an optics unit 12.

The radiation reflected by the specimen 18 is directed toward a detector20 by the optics unit 12.

The optics unit comprises lenses 22, 24 through which the radiation fromthe laser 10 is directed onto a beam splitter 26. From the beam splitter26, the radiation is reflected toward the specimen 18. Through amicroscope objective 28 arranged between the beam splitter 26 and thespecimen 18, the radiation is focused into the specimen 18. Theradiation reflected by the specimen 18 reaches the beam splitter 26again through the microscope objective 28. The radiation given off bythe specimen is let through and directed toward a focusing lens 30 bythe beam splitter 26. Upon detecting fluorescent light, a dichromaticbeam splitter (long-pass) is used. It may also be a semipermeable mirror(not selective as to wavelength).

Through the focusing lens 30, the radiation is focused into the opening32 of an aperture diaphragm 34. After the radiation has passed theaperture diaphragm 34, it impinges onto a detector provided in thedetector unit 20.

The focusing lens 30 is displaceable in the direction of the arrow 36,i.e., in X-direction, in the direction of the arrow 38, i.e., inY-direction as well as in the direction of the arrow 39, i.e., inZ-direction, for adjusting the confocal microscope. Thus, it is possibleto focus the beam path in such a manner by a single adjusting mechanismby which the focusing lens 30 is displaceable in the direction of thethree axes that the focus is arranged in the opening 32 of the aperturediaphragm 34. In the ideal case, the focus thus lies on a central axis40 of the beam path. Further, the central axis 40 is the central axis ofthe aperture opening 32. In the ideal case, the focus is also centeredwith respect to the height of the aperture diaphragm 34 by theadjustment in Z-direction so that an optimum light efficiency isrealized.

In a particular embodiment, the aperture diaphragm 34 is displaceableexclusively in longitudinal direction of the beam path 43 for adjustingthe distance between the focusing lens 30 and the aperture diaphragm 34.Alternately; the aperture diaphragm 34 is rotatable 44 about its centralaxis.

To make the required precision of the adjustment of the focusing lens 30possible, the focusing lens is preferably borne by a linear ballbearing. A good guidance of the focusing lens 30 can also be achieved bya parallelogram guidance that also guarantees a guidance free from playat low efforts. Another possibility of bearing the focusing lensconsists in a three-point bearing that, for example, comprises aV-groove and two adjusting screws, one bearing being configured so as tobe resilient. The three-point bearing has the advantage that it may havea compact configuration and permits a bearing free from play at lowefforts. To this end, the resilient system has to be free from play inthe direction of the optical axis.

In embodiments where the adjustment is not effected via exclusivelyadjusting the focusing lens 30, the beam splitter 26 is adapted to betilted. The beam splitter is tilted about both the X- and the Y-axis. Inthese embodiments, an additional adjustment in the direction of theZ-axis is required which can be realized either by displacing thefocusing lens 30 or the aperture diaphragm 34 together with the detectorunit 20.

Further, a filter 42 may be provided in the beam path. If the filter 42is an exchangeable filter, the course of the beam path can be influencedby the exchange. Thereby, an adjustment becomes necessary. This can beeffected by means of the device according to the invention, preferablyautomatically by adjusting the focusing lens 30.

1. A confocal microscope for single-molecule spectroscopy, comprising: aspecimen holding device for holding a specimen; an illuminating unit; anoptics unit directing radiation produced by the illuminating unit towardthe specimen and directing radiation given off by the specimen toward adetector unit; an aperture diaphragm arranged in a beam path in front ofthe detector unit; and a focusing lens arranged in the beam path infront of the aperture diaphragm, characterized in that the focusing lensis vertically adjustable with respect to the beam path.
 2. A microscopeaccording to claim 1, characterized in that the focusing lens isadditionally adjustable in the direction of the beam path.
 3. Amicroscope according to claim 1, characterized in that the optics unitcomprises a beam splitter for directing the radiation and the beamsplitter is adapted to be tilted to align the beam path with a centralaxis of the opening of the aperture diaphragm.
 4. A microscope accordingto claim 3, characterized in that the beam splitter is exchangeable. 5.A microscope according to claim 1, characterized in that the aperturediaphragm is displaceable exclusively in longitudinal direction of thebeam path for adjusting the distance between the focusing lens and theaperture diaphragm.
 6. A microscope according to claim 1, characterizedin that the aperture diaphragm is rotatable about a central axis of theaperture diaphragm.
 7. A microscope according to claim 1, characterizedin that the aperture diaphragm is stationary.
 8. A microscope accordingto claim 1, characterized in that an opening of the aperture diaphragmis circular or split-shaped.
 9. A microscope according to claim 1,characterized in that an adjusting tolerance with respect to the openingof an aperture diaphragm is smaller than 3 μm.
 10. A microscopeaccording to claim 1, characterized in that the adjusting tolerance withrespect to the opening of the aperture diaphragm is smaller than 2 μm.